Evaluation of TP53 mutations in non-cancerous tissue as novel biomarkers of ovarian cancer risk TP53 is the most mutated gene in human tumors. Surprisingly, recent research has revealed that TP53 mutations are also found at very low frequency in non-cancerous tissues. Whether these mutations increase the risk of future cancer progression is currently unknown. However this possibility is supported by the fact that TP53 mutations in normal tissue are similar to TP53 mutations in cancers: they tend to cluster in hotspots and are mostly deleterious. This indicates an ongoing evolutionary process of positive selection that takes place through life and might result in cancer progression in some individuals. Accordingly, we hypothesize that individuals with an elevated load of somatic TP53 mutations might be at a higher risk of developing cancer. We will test this hypothesis for high grade serous ovarian cancer (HGSOC), a cancer type driven by TP53 mutations and which is typically diagnosed at late stage, causing high mortality. The need for better HGSOC risk biomarkers is especially critical for women with germline mutations in BRCA1 and BRCA2 (BRCA1/2 carriers) who have about 45% and 20% lifelong risk of HGSOC, respectively. Using an ultra-accurate sequencing method called Duplex Sequencing we recently demonstrated that women with HGSOC as well as BRCA1/2 carriers harbor higher frequency of TP53 mutations in blood DNA. Similarly, Pap smear DNA from women with HGSOC contained significantly higher frequency of TP53 cancer-like mutations than Pap smears from women without cancer. These findings support our hypothesis that a constitutionally higher load of TP53 mutant clones might elevate the risk of HGSOC. We propose to further explore these results and their potential utility as cancer biomarkers taking advantage of (1) CRISPR-DS, a Duplex Sequencing based method developed by our group that is more efficient, faster, cheaper, and requires less DNA than the original method; and (2) the University of Washington Gynecologic Oncology Tissue Bank, which has an extensive repository of blood and Pap smear samples collected from women that underwent gynecological surgery for suspected masses or prophylactically due to inherited cancer susceptibility. In Aim 1, we will perform ultra-deep (~3,000x) CRISPR-DS TP53 sequencing of blood DNA from BRCA1/2 carriers and non-carriers with and without HGSOC and we will determine associations between TP53 mutations, HGSOC, and germline BRCA1/2 mutations. For a subset of women, in Aim 2 we will test whether TP53 mutations in Pap smear DNA are also associated with HGSOC and/or germline mutation status and whether the combination of TP53 mutational information from Pap smear and blood has a better cancer predictive value than either test separately. This research breaks ground on the potential biomarker value of somatic TP53 mutations, which were unrecognized until recently due to their very low frequency. CRISPR-DS allows their accurate measurement in a high-throughput manner, thus enabling the development of a translational biomarker that could lead to improved prediction and prevention of HGSOC as well as other cancers driven by TP53 mutations.